Kinases, which constitute a large family of enzymes (>500 in humans), catalyze the transfer of the γ-phosphate of ATP to protein substrates. Reversible phosphorylation plays a paramount role in cell signaling processes and is regulated by kinases and phosphatases. Accordingly, kinases are critical mediators of a myriad of signal transduction processes. Aberrant kinase activity is linked to cancer as well as metabolic, immunological, and nervous system disorders. As a result, kinases have emerged as an important class of drug targets for human disease. However, due to the conserved nature of the active sites of the protein kinase family, it is difficult to obtain selective inhibitors for any one kinase.
There are at least 518 kinases, such as those which catalyze the transfer of the gamma phosphate of ATP to protein and small molecule substrates and are involved in cell signaling processes. Small molecules provide a means for delineating kinase signaling because they are fast acting and dosable. However, because all kinase active sites recognize ATP, it is difficult to develop selective ATP-competitive inhibitors. Several years ago, a chemical genetic strategy for selective kinase inhibition was developed with reversible inhibitors (U.S. Patent Publication No. 2009/0221614). The chemical genetic strategy involves the engineered mutation of a conserved bulky residue in the kinase active site known as “the gatekeeper” to a small residue such as glycine or alanine (See Bishop A C, et al. (1998) Design of allele-specific inhibitors to probe protein kinase signaling. Curr Biol 8(5):257-266; and Bishop A C, et al. (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407(6802):395-401). The engineered active site can then accommodate an inhibitor capable of occupying the newly formed binding pocket. While this strategy has utility, mutation of the gatekeeper residue to a small amino acid may impair the activity of the kinase and the selective inhibition can only be applied to one kinase at a time. In addition, it is sometimes not possible to achieve the desired potency.
It is known in the field that mutations in Leucine-Rich Repeat Kinase 2 (Lrrk-2) can lead to Parkinsons Disease. Also, it is thought that Parkinson's Disease (PD) is caused by uncontrolled apoptosis of dopaminergic neurons. Because inhibition of Lrrk-2 kinase activity can inhibit the apoptotic effects, there is a need to develop inhibitors for Lrrk-2 to provide treatments for Parkinson's Disease.
As such, there is a need in the field to develop kinase gatekeeper residue mutations which do not diminish kinase activity or ATP affinity as well as small molecules which inhibit these kinases. There is also a need to develop effective Lrrk-2 inhig. Surprisingly, the present invention solves these as well as other problems in the field.